Bacterial isolates and their antimicrobial susceptibility profile of superficial and deep-seated skin and soft tissue infections

Abstract Background Skin and soft tissue infections (SSTIs) are caused by microbial invasion of healthy or damaged skin. SSTIs are difficult to manage and contribute to chronicity and emergence of antimicrobial resistance. Objectives To ascertain the prevalence of bacteria causing SSTIs and their antimicrobial susceptibility patterns. Methods A prospective study between November 2020 and May 2021. A total of 447 samples from SSTIs were analyzed. Results A total of 347 samples revealed mono-bacterial growth, of which 67% were male. SSTIs are common among patients aged 21–50 years with the dominance (78%) of gram-negative rods (GNRs). Escherichia coli (36%), Klebsiella spp. (22%), Staphylococcus aureus (16%), and Pseudomonas aeruginosa (11%) were predominant organisms. GNRs were highly resistant (>65%) to ciprofloxacin and trimethoprim–sulfamethoxazole. For injectable antibiotics, the highest resistance was determined against ceftriaxone, and the least resistance was determined against amikacin. Resistance against carbapenem was the highest among P. aeruginosa (53%) and Klebsiella spp. (32%). S. aureus showed the highest resistance against ciprofloxacin, and the least resistance was determined against clindamycin. Of 57 S. aureus isolates, 86% isolates were methicillin-resistant Staphylococcus aureus (MRSA). All isolates of P. aeruginosa and S. aureus were sensitive to polymyxin B and vancomycin, respectively. The prevalence of multidrug-resistant E. coli and Klebsiella spp. was higher among deep-seated SSTIs (dSSTIs). Conclusions The predominant etiology of SSTIs is GNR. Currently, there is very high resistance against oral antibiotics. Antimicrobial resistance against carbapenem has also increased. Moreover, there is a high frequency of MRSA. MDR E. coli and Klebsiella spp. isolates are frequently involved in dSSTIs.

Skin and soft tissue infections (SSTIs) have been defined as invasion of the epidermis, dermis, and subcutaneous tissue by bacteria.The term SSTI covers infections of all anatomical and surgical sites where the integrity of the skin has been breached [1].If SSTIs are just presented with skin redness, they are called mild or superficial SSTIs (sSSTIs), and if present along with systemic signs such as tachycardia, fever, and hypotension, they are classified as deep-seated SSTIs (dSSTIs) [2].The Infectious Diseases Society of America (IDSA) classifies SSTIs into 2 categories: purulent infections (e.g., furuncles, carbuncles, and abscesses) and non-purulent infections (e.g., erysipelas, cellulitis, and necrotizing f asciitis) [1].SSTIs are usually associated with prolonged hospital stay, increased medical cost, and diverse etiology.The management of dSSTIs is challenging and often requires hospitalization, incision, drainage, or debridement [3].Predisposing factors include old age, trauma, cardio-pulmonary or hepatorenal disease, diabetes mellitus, and immunosuppression [4].
Although SSTIs affect all age groups, they are more frequent among men aged <5 years and >65 years, involving the lower leg region [5].Annual outpatient visits for SSTIs are estimated at >14 million/year in the United States [2].Nearly 7%-10% of admitted patients are affected by SSTIs, and they represent the third most common diagnosis after chest pain and asthma in emergencies [6].
Similarities in clinical presentation and low probability of isolating an organism make the diagnosis challenging [5].Treatment decisions are often empirical and based on educational guess, host characteristics, likely pathogens, and local susceptibility patterns [5].Treatments are frequently topical, oral, intravenous, or surgeries [11].Oral formulations are preferred to avoid higher cost and adverse effects of intravenous administration.Intravenous treatment is reserved for inpatients, for life-threatening dSSTIs, or for those who are unable to absorb oral formulations or fail to achieve adequate concentration at the infection site [12].
The increase in antibiotic resistance and its rapid spread among pathogens has posed a threat to public health worldwide [10].The growing prevalence of multidrug-resistant pathogens like MRSA and carbapenem-resistant enterobacteriaceae (CRE) is a global concern [9].Although few studies from Pakistan report the incidence rate and antimicrobial susceptibility profile of Staphylococcus aureus (S. aureus) causing SSTIs, the data for other microbes are limited [11,[13][14][15][16][17].

Methods
Ethical approval was obtained from the Ethical Review Committee of the Sindh Institute of Urology and Transplantation (approval No. SIUT-ERC-2020/A-244).Because this was a laboratory-based study, the ERC had exempted this research from obtaining informed consent from patients.

Study design
A prospective laboratory-based study in the Department of Microbiology, Sindh Institute of Urology and Transplantation (SIUT), Pakistan, conducted from November 2020 to May 2021.

Study population
A total of 447 patients of all ages and both sexes attending outpatient clinics or admitted in wards with SSTIs were included.A physician recorded demographic characteristics and underlying cause and stratified the SSTIs into sSSTIs and dSSTIs.

Inclusion criteria
Mono-bacterial growth either gram-positive cocci (GPC) or GNRs.We used the STROBE cross-sectional checklist to report our results.

Sample collection and processing
Pus swabs, discharge/secretion, exudates, aspirate, or tissue samples from SSTIs were inoculated on the chocolate, blood, and MacConkey agar (Oxoid Ltd, Hampshire, UK).
Susceptibility to polymyxin B and vancomycin was determined using the microbroth dilution method and E test, respectively.Polymyxin B MIC interpretation was done as intermediate = 2 µg/mL and resistant = 4 µg/mL.A cefoxitin 30 µg disk was used to determine MRSA.The SCF zone diameter of ≤15 mm was interpreted as resistant, 16-20 mm as intermediate, and ≥21 mm as sensitive [18].The Clinical Laboratory Standard Institute (CLSI) M100 guidelines were used to interpret the results [19].

Multidrug resistance
Multidrug resistance (MDR) is defined as bacterial resistance to at least 1 agent in 3 or more antimicrobial classes [7].

Data analysis
Data analysis was performed using MS excel, SPSS version 20, and GraphPad prism 9.3.1.

Results
A total of 447 specimens from 447 SSTI patients ranging in age from 1 to 78 years, with mean age of 33.76 ± 21.27 years, were included in this study.Of these, mono-microbial growth was isolated from 347 (78%) patients, of which 61% were inpatients and 39% were outpatients.
Of 347 patients, 67% were male and 33% were female.Most of the patients (157 (45%)) were in the 21-50 years age group, 26% were older than 51 years, and 18% were in the 1-20 years age group.The age data were missing for 38 (11%) patients (Table 1).
The prevalence of E. coli, Klebsiella spp., Pseudomonas aeruginosa and S. aureus was significantly higher among dSSTIs (Figure 1).
For injectable antibiotics, the least level of resistance among each GNR was determined against amikacin, and the highest was determined against ceftriaxone.P. aeruginosa and Klebsiella spp.were highly resistant to SCF (42% and 49% respectively) and TZP (34% and 43%,).E. coli showed the highest resistance against SCF (30%) and TZP (29%), and the least resistance against amikacin (9%).None of the P. aeruginosa isolates showed resistance against polymyxin B.
The MDR prevalence of E. coli, Klebsiella spp., P. aeruginosa, and S. aureus was 70%, 75%, 58%, and 73% respectively.Overall, the prevalence of E. coli and Klebsiella MDR among dSSTIs was higher than that in P. aeruginosa and S. aureus, which was higher among sSSTIs (Table 3).

Discussion
In this study, mono-bacteria were isolated from 78% of patients, of which 67% were males.Similar results have been reported from Pakistan and Ethiopia with the single pathogen isolation (77% and 82%, respectively) with male dominance [11,7].In the present study, the incidence of SSTIs was higher (45%) among patients in the 21-50 years age group; this is in agreement with previous studies that report higher prevalence in the 15-44 years age group [11,7].Moreover, the rate of SSTIs was higher among inpatients (61%) in our study, which is analogous to a previous study (72.8%) [7].In this study, dSSTIs were more common (64%), among which abscess and surgical sites were the major sites (94%), whereas wound was the most common site (66%) for sSSTIs.This was in agreement with a similar study from Pakistan, where abscess was the most common site [11].Chronic kidney disease and diabetes mellitus were major underlying conditions (64%) in our patients.A previous study has reported diabetes mellitus, chronic kidney disease, and heart failure as the most common underlying medical conditions [8].
In the current study, the GNR/GPC ratio was 4:1.Furthermore, the prevalence rate of GNR was higher (79%) than reported in the studies conducted in India, Ethiopia, and Greece (57%, 57%, and 55%, respectively) [7][8][9].The most frequent GNRs in this study were E. coli (36%), followed by Klebsiella spp.(22%) and P. aeruginosa (11%).The results are in concordance with a study from Pakistan that reported E. coli and P. aeruginosa from complicated SSTIs as the most frequent GNRs.Another study from India reported Asian Biomed (Res Rev News) 2023; 17(2):55-63 P. aeruginosa (21%) and E. coli (17.5%) as predominant GNRs [9].This deviance in the isolation may be due to geographical variation or study design.S. aureus was the predominant (16%) GPC found in this study, which is in agreement with studies published from Pakistan and Nepal [11,20].
For injectable antibiotics, only a least number of GNRs were resistant against amikacin and a highest number were resistant against ceftriaxone in our study.We report >79% of E. coli and Klebsiella spp.were resistant against ceftriaxone.A similar high resistance rate of >80% of Klebsiella spp.and E. coli against third-generation cephalosporins has been reported [9,11].E. coli was found the least resistant against amikacin (9%) in this study., which is in contrast to other studies where they have ascertained a very high resistance rate among E. coli against amikacin (63%-80%).For Klebsiella spp., resistance against amikacin was 38% in our study, which is similar to the resistance reported in a study from India (40%) and higher than that reported in a study from Pakistan (25%) [9,11].This indicates that resistance against amikacin among Klebsiella spp. is increasing in Pakistan.In this study, 39% of P. aeruginosa demonstrated resistance against amikacin, which is lower than that reported in studies from Pakistan (66.6%) and India (47.8%) [9,11].
In contrast to a previous study from Pakistan where resistance against TZP among Klebsiella spp., P. aeruginosa, and E. coli was reported 12.5%, 6%, and 2%, respectively.Our findings demonstrate a much higher rate of resistance, i.e., 43%, 34%, and 29%, respectively.This significant increase in resistance in our isolates may be due to the overuse of antibiotics in our hospital.However, our results are similar to those of a study from India where high resistance prevalence among E. coli (63%) and Klebsiella spp.(60%) against TZP has been reported [9,11].In our study, the rate of resistance among P. aeruginosa, Klebsiella spp., and E. coli to SCF was 42%, 49%, and 30%, respectively.
In this study, a very high level of resistance against carbapenem was found among P. aeruginosa (53%), Klebsiella spp.(32%), and E. coli (18%) in contrast to a previous studies from Pakistan and India where low resistance among Klebsiella spp.(0% and 0%, respectively), P. aeruginosa (8.4% and 8.6%, respectively), and E. coli (1.7 and 2.6%, respectively) has been reported.Another report form the USA and Europe also reported all E. coli isolates were sensitive to imipenem [21].This high level of resistance against carbapenem in our region may be due to the increasing expression of extended spectrum beta-lactamases, the use of self-medication, and indiscriminate use of antibiotics.In our study, all isolates of P. aeruginosa and S. aureus were sensitive to polymyxin B and vancomycin, respectively.This may be accredited to the presence of intrinsic genes among these isolates [22].
We noticed differences in the resistance pattern of isolates from dSSTIs and sSSTIs.A comparatively high number of E. coli and Klebsiella spp.isolates were resistant from dSSTIs against oral, injectable, and carbapenem antibiotics, whereas S. aureus and P. aeruginosa isolated from sSSTIs were more resistant.
The cumulative resistance rate of bacteria from hospitalized patients was higher in our study, indicating the circulation of highly resistant bacteria, particularly MDR E. coli, Klebsiella spp., and S. aureus within the hospital environment, which alarms for continuous monitoring and isolation of patients from immunocompromised and health care workers.This was in agreement with the previous reports where a higher proportion of antimicrobial resistance among inpatients has been reported [23].
The predominant GPC in our study was S. aureus (78%).Notably, a high prevalence of MRSA (86%) was ascertained in our study population compared to that in previous studies from Pakistan (53%, 36.1%, and 42%) [24][25][26].A meta-analysis of 37 studies from China has reported the pooled prevalence of MRSA (21.2%) [27].This strikingly high frequency of MRSA might be attributed to the frequent use of empirical treatment for SSTIs, a paradigm shift in the daily life style due to the COVID-19 pandemic, or a typical public hospital with overuse of intravenous drugs.In our study, 74% of S. aureus were multidrug-resistant, which is lower than that reported in a previous study from Ethiopia 94.8% [20].
The high prevalence of SSTIs was found among males in the 21-50 years age group.The predominant isolates were E. coli, Klebsiella spp., P. aeruginosa, and S. aureus.More than 50% of all GNRs were resistant to each oral antibiotic.For intravenous antibiotics, the least resistance was determined against amikacin, and the highest was determined against ceftriaxone.For carbapenem, P. aeruginosa showed the highest resistance (53%).S. aureus showed the highest resistance against ciprofloxacin, and the least resistance against clindamycin.MDR E. coli and Klebsiella spp.isolates were found to be predominant among dSSTIs.We recommend continuous surveillance for appropriate antibiotic usage and for preventing the emergence of MDR pathogens.

Table 2 .
Antimicrobial resistance pattern of common bacteria isolated from sSSTIs and dSSTIs against oral, injectable, and carbapenem antibiotics Bacterial